The obligate intracellular parasite Toxoplasma can infect all nucleated cells and resides within a parasitophorous vacuole where it must salvage nutrients from host cells. Endocytosis is therefore an important mechanism for nutrient acquisition and represents a logical and potential target for antiparasitic therapy and drug delivery. Surprisingly, endocytosis is relatively uncharacterized in Toxoplasma, or in the related malarial parasite Plasmodium. This proposal aims to provide the groundwork for understanding the endocytic pathway in T. gondii. First, the endocytic cascade will be mapped by pulse chase internalization studies of fluid-phase tracers and biotinylated surface proteins using confocal or cytochemical EM. Second, the mechanism controlling the endocytic machinery will be examined by kinetic studies of endocytosis in intact and permeabilized cells, by pharmacological manipulation of cytoskeletal and signal transduction components. Third, the universal tyrosine-based internalization signal will be evaluated in T. gondii by the use of foreign endocytic reporters. These reporters contain an E. coli beta-lactamase ectodomain and the transmembrane and cytoplasmic tail of the LDL receptor. Introduction of well-defined mutation in the internalization signal of the LDL-R cytoplasmic tail will be used to probe the endocytic machinery of T. gondii without the requirement to identify individual components in the pathway. These experiments will be an important step for future toxin delivery or targeted-disruption of the endocytic pathway in apicomplexan parasites.